Medical diagnostics are increasingly incorporating nucleic acid-based assays. Such assays include, e.g., qualitative measurements of the absence or presence of a nucleic acid (i.e., various forms of DNA and RNA), quantitative measurements of the amount of a nucleic acid in a biological sample, and determinations of the physical characteristics or modification state of a nucleic acid. However, biological materials are often contaminated with substances that damage nucleic acids during storage or inhibit enzymatic manipulation of the nucleic acids, e.g., digestion with restriction enzymes or amplification by polymerase chain reaction (PCR). Therefore, to use the nucleic acids isolated from certain biological materials for further analysis, it is important that the amounts of these inhibiting substances should be minimized, i.e., they should be present only in very low concentrations or in some cases should be eliminated entirely from the sample.
Purifying nucleic acids from biological samples commonly uses a combination of different purification steps such as protease treatment, phenol/chloroform extraction, binding of nucleic acids to silica in the presence of chaotropic salts, gel filtration, anion exchange chromatography, and the use of cationic or anionic detergents. Nucleic acids isolated from biological samples by these methods are, however, often unstable and behave problematically in subsequent enzymatic reactions such as PCR, for example. Many of these problems arise due to the presence of substances that are co-isolated with the nucleic acids and that damage them and inhibit enzymatic reactions. Examples of these inhibitors from biological sources include hemoglobin and its metabolites, bile acids and bile acid derivatives, and polysaccharides.
These substances are especially problematic for assays directed to measuring rare events, e.g., rare mutations or other rarely occurring genetic modifications. Assays designed to detect low-incidence events are vulnerable to inhibitors for at least two related reasons. Highly sensitive assays designed to detect extremely small amounts of nucleic acids may be compromised by trace amounts of inhibitors. Also, such assays require large amounts of a nucleic acid for analysis and thus often the assays will also include substantial amounts of the co-purified inhibitors.
Others have attempted to solve this problem using a variety of techniques. Most conventional methods first employ a total nucleic acid precipitation step followed by inhibitor removal or inactivation using proprietary adsorption resins and optimized buffers. These methods, though, are not applicable to all samples and assays because, while some inhibitors are soluble and thus can be separated from the nucleic acid preparation in these methods, a large number of inhibitors co-precipitate with the nucleic acid preparation.
Moreover, many highly sensitive assays require maximizing the volume of isolated nucleic acids that comprise the assay volume, e.g., by using a large sample as input and concentrating the nucleic acid sample into a small volume. Conventional solutions, though, do not function adequately when scaled up for processing large samples. They either cannot accommodate large samples or are not sufficiently effective on the large samples. When even relatively small amounts of inhibitors remain, only a very limited amount of the isolated nucleic acid can be used in subsequent assays without inhibiting a subsequent assay or other reaction. Consequently, the amount of material that can be added to the assay is insufficient to reach the sensitivity limits of some assays.